Our hypothesis contends that manufactured carbon nanoparticles (fullerenes, single-walled nanotubes, and multi-walled nanotubes), when exposed to barrier epithelial (bronchiolar, intestinal, and kidney) cells, exert biological effects directed at the membrane and associated cytoskeletal proteins that alter cell function. Specifically, vesicular trafficking and the cytoskeletal components and events associated with vesicular trafficking are likely targets. Alterations in membrane-associated proteins will also alter or dysregulate electrogenic transport mechanisms. We propose that these effects are directly related to the size and physico-chemical character of the carbon nanoparticles, and, at some specific dose, are likely to be injurious to the cells. They thus may pose a significant health hazard. To study this, we plan to expose the three cell types to the three carbon nanomaterials mentioned above, in vitro, using a dose-response design spanning 3 orders of magnitude for a duration of 24 and 48 hrs. After exposure, we will examine differential protein expression using quantitative mass spectrometry and two-dimensional electrophoresis. We will also characterize alterations in protein post-translatiohal modification using those proteomic techniques. We will examine the functional effects of the exposures by measuring ion transport and transepithelial electrical resistance and the potential changes induced by nanoparticle exposure for the response to normal, endogenous regulators. In addition, we will quantify irritation/injury/toxicity using various measurements of cell cytokine secretion, reactive-oxygen species formation, and cell viability. An important aspect of this project is to accurately assess the elemental composition, particle number, particle size distribution, particle shape, and surface area of the primary particle and agglomerated material, as well as the culture-media agglomeration/de-agglomeration status. The results of these studies will provide important new information regarding the effect that different carbon nanoparticles have on barrier epithelial cell global protein expression. In addition, the studies will correlate the changes in protein expression with changes in cellular function at both toxic and subtoxic levels.